Abstract
Purpose :
Corneal epithelium (CE) forms the protective layer of cornea against ultraviolet (UV) radiation damage. Continuous exposure of CE to UV radiation in high UV index regions causes DNA damage and genetic instability resulting in corneal damage. Previously we demonstrated that Krüppel-like factor 4 (KLF4) plays a crucial role in CE homeostasis. Here we examined the role of KLF4 in CE genetic stability and ferroptosis in response to UV exposure.
Methods :
Control human corneal limbal epithelial (HCLE) cells and those overexpressing KLF4 (HCLE-KLF4) were exposed to 100 J/m2 UV-B radiation or treated with increasing amounts (1.6 -100 mM) of hydrogen peroxide (H2O2). The resulting cellular morphology was evaluated by phase contrast microscopy, cell viability by crystal violet staining, selected mRNA and protein expression by qRTPCR, immunoblots and immunofluorescent staining, and reactive oxygen species levels quantified by H2DCFDA using spectrophotometry.
Results :
Exposure of HCLE cells to UV-B radiation resulted in apoptotic changes; bleb formation and extensive cell death coupled with increased expression of antioxidant genes-glutathione peroxidase 4 (GPX4), proinflammatory IL6, cell cycle inhibtors-p53 and p21, and decreased expression of DNA damage repair gene- ATM. UV-B-exposed HCLE-KLF4 cells displayed increased ATM expression, and less accumulation of H2AX at the nascent DSB site, decreased levels of phospho-p53 and reactive oxygen species (ROS) compared with the control HCLE cells. Both HCLE and HCLE-KLF4 cells showed decreased ROS production and increased cell viability after treatment with Ferrostatin-1 (inhibitor of ferroptosis), in comparison to the untreated cells.
Conclusions :
Based on these results, we conclude that KLF4 is essential for maintaining CE cells' genetic stability by regulating ROS levels and DNA damage-associated molecules. We also demonstrated that inhibition of UV-induced ferroptosis promotes CE cell survival.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.